Mechanism Of S1P Secreted By Endothelial Cells Promoting Migration Of Colorectal Cancer By Binding To GPR63 And Activating JAK2-STAT3 Signaling Pathway

Background and purposeColorectal cancer ranks third in the global incidence of cancer,and its mortality is second of the malignant tumors.Colorectal cancer metastasis is the main cause of poor quality of life and death in patients with colorectal cancer.Cancer metastasis involves a series of complex interactions between tumor cells and microenvironment that influence its biological effectiveness and facilitate tumor cell arrest to distant organs.Successful metastatic growth depends on the ability of cancer cells to respond to appropriate microenvironments at each step in the metastatic cascade,and the microenvironment plays a guiding role in tumors metastasis.G protein-coupled receptor 63(GPR63)is a gene related to colorectal cancer migration,which is sequenced and analyzed by two groups of colorectal cancer cells with different migration ability.GPR63 is a member of G protein-couples receptors(GPCRs).GPCRs,also known as the 7-transmembrane receptor family,because of its seven transmembrane helix subunits,receiving extracellular signals such as odors,hormones,and neurotransmitters,etc,control a variety of life activities of cells,such as proliferation,differentiation,chemotaxis and intercellular signaling,which is one of the main directions of clinical drug research.GPR63,a transmembrane receptor,is activated with SIP,according to reports in the literature,which suggests that Sphingosine-1-phosphate(SIP)and dioleoylphosphatidic acid(LPA)are low affinity agonists of GPR63.SIP,secreted by vascular endothelial cells,red blood cells and platelets,is a potent bioactive sphingolipid metabolite that regulates a variety of cellular processes,including cell growth,survival,differentiation,lymphocyte transport,vascular integrity,and cytokines and chemotaxis factors generation,etc.So far,the mechanism by which SIP affects GPR63 to promote tumor metastasis is unclear.This paper aims to reveal the mechanism by which GPR63 is affected by tumor microenvironment to promote the migration of tumor cells to blood vessels.MethodsThe expression of GPR63 in paraffin specimens of colorectal cancer tissues was detected by immunohistochemistry.The biological functions of GPR63 in colorectal cancer cells were explored by a series of in vitro experiments,such as CCK8,migration assay,and sphere formation assay.Migration experiments and Western Blot were used to verify the effect of SIP on GPR63.The ability of tumor cells to migrate to blood vessels was examined by trans-vascular endothelial migration assay.The interaction of GPR63 and Src was detected by immuno-convo-focusing,co-immunoprecipitation and Src interference assay.The signal pathway affected by GPR63 is predicted by GSEA and verified by Western-Blot.Results(1)Immunohistochemistry was used to detect the expression of GPR63 on paraffin sections of colorectal cancer tissues.The results showed that GPR63 was negatively expressed in normal intestinal cells,but positively expressed in cancer tissues which were located in cytoplasm and cell membrane.Clinical pathological information found that high expression of GPR63 was positively correlated with lymph node metastasis and distant metastasis of colorectal cancer.(2)With the stimulation of SIP,GPR63 promotes the migration and invasion of colorectal cancer cells and enhance the stemness.The migration ability of colorectal cancer cells is enhanced by SIP stimulation,and inhibited when FTY720 is blocked by S1R.In sphere formation assays,high expression of GPR63 enhanced the stermness of colorectal cancer.The difference in GPR63 expression in CCK8 and plate cloning experiments had no significant effect on the proliferation of colorectal cancer cells.In the experiment,high expression of GPR63 promoted the transvascular migration of colorectal cancer cells.Migration assays showed that high expression of GPR63 did not affect the migration of colorectal cancer cells without SIP stimulation.(3)Co-immunoprecipitation experiments showed that GPR63 and Src were combined with each other and the fluorescence colocalization experiments showed that GPR63 and Src exist spatial colocalization.After interfering with Src expression,the migration ability of tumor cells was significantly reduced.High expression of GPR63 increased Src and stat3 phosphorylation and the expression of CD44 and CD133.While HUVECs cells(human umbilical vein endothelial cells)cocultured with LoVo cells,LoVo cell surface was activated by S1P secreted by vascular endothelial cells.GPR63 increases the level of intracellular Src phosphorylation and activates the JAK2-STAT3 signaling pathway,which in turn increases the migration and stemness of colorectal cancer cells.Conclusion1.GPR63 is expressed in cancer cells of colorectal cancer and is positively correlated with lymph node metastasis and distant metastasis;2.SIP secreted by endothelial cells induce promotes the migration of GPR63 with high expression of colorectal cancer cells and enhances their stemness;3.With S1P stimulation,GPR63 binds to Src to activate downstream JAK2-STAT3 signaling pathway,enhancing the migration and invasion ability and stemness of colorectal cancer cells..